Project Summary The X-chromosome is enriched with immunity-related genes, therefore X-linked genes need to be regulated to prevent abnormal expression. Females use X-chromosome Inactivation (XCI) to equalize X-linked gene expression, where one X maintained transcriptionally silent by continuous expression of the long noncoding RNA Xist and enrichment of heterochromatin modifications. We have recently discovered that female lymphocytes have a unique and dynamic mechanism to maintain XCI, unlike other somatic cells. Resting mature B cells lack Xist RNA and heterochromatin marks on the Xi, and these modifications return to the Xi through a YY1-mediated mechanism upon stimulation. Our in-press work indicates that preventing Xist RNA localization to the Xi by conditional knock-out of YY1 impairs heterochromatin enrichment on the Xi, and increases X-linked expression. In preliminary work, we found that B-cell specific deletion of one Xist allele (mb1CRE XistCKO/+) dramatically reduced Xist localization patterns over the Xi, reduced heterochromatin enrichment, and increased expression of specific X-linked genes. Moreover, this increase in X-linked gene expression was accompanied by increased antibodies to double-stranded DNA, a hallmark of autoimmunity. We hypothesize that Xist RNA localization to Xi is required to keep Xi at the nuclear periphery to maintain transcriptional repression in activated B cells, and that failure to localize Xist RNA disrupts Xi nuclear organization and perturbs X-linked gene expression, with consequent predisposition to autoimmunity. We will test our hypothesis with the following aims: (1) Do temporal and sequence-specific occupancy of YY1, Xist RNA, and heterochromatin marks on the Xi maintain transcriptional repression in activated splenic B cells? (2) Do chromosome structural proteins cooperate with YY1 to localize Xist RNA within Xi territory at the nuclear periphery for transcriptional repression in activated splenic B cells? (3) Are Cxcr3, Itm2a, Syn1 and Cfp overexpressed in lupus mouse models and does increased dosage predispose to autoimmunity? IMPACT: The results from these experiments will yield fundamental insight about the lymphocyte-specific mechanisms that regulate expression from the Xi, and will advance our understanding of the female-bias underlying B cell mediated autoimmune disorders.